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1、1基因（Gal-1）在大肠杆菌中的融合表达与纯化ChineseJournalofVeterinaryMedicine中国兽医杂志2006年(第42卷)第9期鸡I3一防御素一1基因(Gal一1)在大肠杆菌中的融合表达与纯化张辉华,毕英佐,曹永长,马静云,杜礼琴(1.华南农业大学动物科技学院,广东广州510642;2.佛山科学技术学院生命科学院,广东佛山528231)摘要:内源性抗菌多肽防御素是机体先天性免疫系统的重要组成部分,可帮助机体防御外界病原微生物的入侵.鸡防御素-I(Gallinacin-1,Ga
2、l～1)对许多致病菌都具有杀菌作用,具有很好的研究开发价值.本研究利用PCR技术从重组质粒pGEM-TEasy-gal-1中克隆出Gal-1基因成熟肽区,将该基因插入原核表达载体pET一32a(+)中,构建重组质粒pET一32a(+)一gal-I,然后用重组质粒转化大肠杆菌BL-21.挑选阳性克隆菌,用浓度为0.5mmol/L的IPTG进行诱导表达.SDS—PAGE电泳显示在目标位置约25kD处出现了条带并用WersternBlot进行了鉴定.表明Gal一1基因在大肠杆菌以融合形式得到了表达.经灰度扫描
3、显示重组蛋白表达量占细菌总蛋白的15.54.用5ONi—NTA纯化树脂进行层析纯化,在洗脱液E中出现单一条带.Gal一1多肽在pET一32a(+)质粒表达体系中成功表达为鸡防御素多肽下一步的研究奠定基础.关麓词:鸡防御素一1(Gal一1);融合表达;蛋白纯化中图分类号:Q786文献标识码:A文章编号:0529—6005(2006)09—0009—0SThefusionexpressionandpurificationofchickenp—defensin一1(Gal一1)geneinEscherichi
4、acoliZHANGHui—hua",BIYing—ZUO¨,CAOYong—chang,MAJing—yun,DULi—qin(1.CollegeofAnimalScience,SouthChinaAgriculturalUniversity,Guangzhou510642,China;2.CollegeofBiology,FoshanScienceandTechnologyCollege,Foshan528231,China)Abstract:Endogenousantimicrobialpepti
5、dedefensinsareessentialcomponentsofinnateimmunity.DefensinsaredistributedthroughoutthehosttOcontrolinvadingmicro—organisms.Chiekenbeta—defensin一1(Gallinacin-1,Gal一1)hasantimicrobialactivityagainstpotentiallypathogensandisworthwhilebeinginvestigatedanddev
6、eloped.ThematurefragmentofGal一1genewasamplifiedfromtheplasmidofpGEM—TEasy-gal-1,andtheninsertedintotheEcoRIandBamHIenzyme—cuttingsitesofpET一32a(+)plasmid.TherecombinantvectorsnamedpET一32a(+)一gal一1weretransformedintothecompetenteellE.colBL一21.Thepositivec
7、loneswereculturedandinducedtoexpresstargetproteinbyadditionofO.5mmolIPTGinLA.Expressionproductswithmolecularweightofapproximately25kDawereexaminedbvSDS—PAGEandWesternBlot,whichshowedthatgal一1genewasexpressedintheformoffusionprotein.Densitometricscanninga
8、nalysisdemonstratedthatthefusionproteinaccountedforabout15.5ofthetotalbacterialprotein.Theexpressedfusionproteinswerepurifiedwith5ONi—NTAresinaffinitychromatography.Highlypurifiedproteinswereachievedintheeluate.Furtherstud
